PROJECT SUMMARY TMJ osteoarthritis (OA) poses a major clinical problem, but there are no therapies that promote TMJ regeneration. Therapeutically exploiting resident stem cells represents a stem cell-based strategy for tissue regeneration. We have identified TMJ fibrocartilage stem cells (FCSCs) localized within the TMJ condyle superficial zone. Transplanted FCSCs engraft, self-organize and regenerate cartilage, fat and vascularized bone. FCSCs are heterogeneous, yet markers critical for studying signals regulating FCSCs are unknown. We found that Prg4+, Lgr5+ and Prg4+Lgr5+ cells represent FCSC populations in rodents and pigs. We discovered that Wnt/?Catenin signaling is critical for FCSC fate specification by inducing proliferation and inhibiting differentiation of FCSCs. However, over-active Wnt/?Catenin is associated with TMJ OA. We developed two TMJ disease models in mice and pigs and discovered that TMJ OA is correlated with increased ?Catenin and apoptosis mediated FCSC depletion. These data suggest inhibition of Wnt/?Catenin may serve as a therapeutic regenerative strategy for the treatment of TMJ OA. In fact, we show that therapeutic application of a canonical Wnt inhibitor repairs TMJ in a rabbit TMJ injury model. Based on our published work and preliminary data, we hypothesize that Prg4+, Lgr5+ and Prg4+Lgr5+ are FCSCs and Wnt/?Catenin inhibition in FCSCs regenerates TMJ. We will test our hypothesis using three specific aims: 1) Define FCSC populations critical for TMJ development and homeostasis; 2) Determine the role of Wnt/?Catenin signaling in regulating FCSC fate; 3) Target resident FCSCs to regenerate cartilage in a porcine TMJ injury model. Using mouse genetics, we will characterize FCSCs and delineate the role of Wnt/?Catenin signaling in regulating FCSC fate. We will optimize a new, clinically relevant large animal model and test Wnt inhibition in FCSCs as a potential therapy for TMJ OA. Taken together, these novel studies will contribute towards the development of a non- surgical, minimally invasive stem cell-based therapy for TMJ regeneration.